Process for the purification of glycoproteins

Abstract

The present invention relates to a process for the purification of a glycoprotein comprising subjecting a liquid containing said glycoprotein to the steps of: a) reverse phase chromatography, b) size exclusion chromatography, and c) hydrophobic interaction chromatography. Also provided is a manufacturing process for producing a glycoprotein of interest.

The invention claimed is: 
     
       1. A process for the purification of a gonadotropin comprising subjecting a biological sample containing the gonadotropin to the steps of:
 a) reverse phase chromatography as a first chromatographic step, 
 b) size exclusion chromatography, and 
 c) hydrophobic interaction chromatography; 
 wherein the steps are performed in the sequence of reverse phase chromatography, size exclusion chromatography, and hydrophobic interaction chromatography. 
 
     
     
       2. The process according to  claim 1 , wherein in reverse phase chromatography step (a), the elution buffer contains an organic solvent. 
     
     
       3. The process according to  claim 1 , wherein in size exclusion chromatography step (b), a buffer exchange is performed. 
     
     
       4. The process according to  claim 1 , further comprising one or more steps selected from the group consisting of chromatography steps, filtration steps and virus inactivation steps. 
     
     
       5. The process according to  claim 1 , further comprising one or more steps elected from the group consisting of ion exchange chromatography, affinity chromatography, diafiltration, ultrafiltration, nanofiltration and virus inactivation. 
     
     
       6. The process according to  claim 1 , further comprising an anion exchange chromatography step. 
     
     
       7. The process according to  claim 6 , wherein the anion exchange chromatography step is carried out subsequent to size exclusion chromatography step b). 
     
     
       8. The process according to  claim 6 , wherein at least one salt-containing elution buffer is used in the anion exchange chromatography step. 
     
     
       9. The process according to  claim 6 , wherein different charged isoforms of the glycoprotein are separated. 
     
     
       10. The process according to  claim 1 , wherein the gonadotropin is selected from the group consisting of follicle-stimulating hormone (FSH), chorionic gonadotropin (CG), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH). 
     
     
       11. The process according to  claim 1 , wherein the gonadotropin is produced recombinantly. 
     
     
       12. A process for the purification of a gonadotropin comprising subjecting a biological sample containing the gonadotropin to the sequential steps of:
 (0) Ultrafiltration; 
 (1) Reverse phase chromatography as a first chromatographic step; 
 (1a) optionally Ultrafiltration; 
 (2) Size exclusion chromatography; 
 (3) Anion-exchange chromatography; 
 (4) Hydrophobic interaction chromatography; 
 (5) Ultrafiltration and/or diafiltration; 
 (6) Nanofiltration. 
 
     
     
       13. A process for manufacturing a gonadotropin of interest, comprising the following steps:
 i) recombinantly expressing the gonadotropin; 
 ii) purifying the gonadotropin by subjecting a biological sample containing the gonadotropin at least to the steps of: 
 a) reverse phase chromatography as a first chromatographic step, 
 b) size exclusion chromatography, and 
 c) hydrophobic interaction chromatography; 
 wherein the steps are performed in the sequence of reverse phase chromatography, size exclusion chromatography, and hydrophobic interaction chromatography. 
 
     
     
       14. The manufacturing process according to  claim 13 , further comprising one or more of the following steps:
 (a) ion exchange chromatography; 
 (b) anion exchanged chromatography; 
 (c) affinity chromatography; 
 (d) ultrafiltration; 
 (e) diafiltration; 
 (f) nanofiltration; and 
 (g) virus inactivation. 
 
     
     
       15. The manufacturing process according to  claim 13 , wherein the gonadotropin is selected from the group consisting of FSH, CG, LH and TSH. 
     
     
       16. The process according to  claim 2 , wherein the organic solvent is isopropanol. 
     
     
       17. The process according to  claim 10 , wherein the gonadotropin is FSH. 
     
     
       18. The process according to  claim 1 , further comprising one or more steps selected from the group consisting of anion exchange chromatography, cation exchange chromatography, dye affinity chromatography, immune affinity chromatography, lectin affinity chromatography and perborate affinity chromatography. 
     
     
       19. The process according  claim 1 , additionally comprising a chromatofocusing step. 
     
     
       20. The process according to  claim 1 , wherein the process
 (i) does not comprise a buffer exchange step between the reverse phase chromatography and the size exclusion chromatography; and/or 
 (ii) does not comprise a buffer exchange step between the size exclusion chromatography and the anion-exchange chromatography or the hydrophobic interaction chromatography; and/or 
 (iii) does not comprise an immunoaffinity chromatography and/or a cation exchange chromatography; 
 (iv) comprises only the three chromatographic steps reverse phase chromatography, size exclusion chromatography and hydrophobic interaction chromatography; or comprises only the four chromatographic steps reverse phase chromatography, size exclusion chromatography, anion exchange chromatography or chromatofocusing step, and hydrophobic interaction chromatography. 
 
     
     
       21. The process according to  claim 1 , wherein the biological sample is selected from the group consisting of cell culture medium, cell lysates, and natural source liquids. 
     
     
       22. The process according to  claim 12 , wherein the biological sample is selected from the group consisting of cell culture medium, cell lysates, and natural source liquids. 
     
     
       23. The process according to  claim 13 , wherein the biological sample is selected from the group consisting of cell culture medium, cell lysates, and natural source liquids.